ANALYSIS OF TRANSCRIPTION AND ESTROGEN INSENSITIVITY IN THE FEMALE MOUSE AFTER TARGETED DISRUPTION OF THE ESTROGEN-RECEPTOR GENE

We employed homologous recombination in mouse embryonic stem cells to disrupt the estrogen receptor (ER) gene. Subsequently generated mice t hat are homozygous for the gene disruption, termed ERKO, possess no de monstrable wild-type ER by Western blot analysis. However, the presenc e of residual high affinity binding, as detected by [H-3]estradiol bin ding assays and sucrose gradients in uterine extracts from ERKO female s prompted further investigation of transcription and translation prod ucts from the disrupted ER gene. Analysis of ERKO uterine messenger RN A (mRNA) by reverse transcriptase-polymerase chain reaction demonstrat ed that although no full-length wild-type ER mRNA was present, two sma ller transcripts, labeled E1 and E2, were identified and partially seq uenced. Both ERKO transcripts are splicing variants that result in the disrupting NEO sequence being partially or completely removed from th e mRNA. In the ERKO-ES variant, this results in a frame shift and the creation of at least two stop codons downstream. In the ERKO-E1 varian t, the ER reading frame is preserved and encodes for a smaller mutant ER that could be the source of the residual estradiol binding. When th is mutant form is overexpressed and characterized in vitro, it results in a smaller protein of the predicted size that possesses significant ly reduced estrogen-dependent transcriptional activity compared with t hat of the wild-type ER. Despite residual amounts of an impaired ER va riant, estrogen insensitivity in the female ERKOs was confirmed by the failure of estrogen treatment to induce known uterine markers of estr ogen action, such as increased DNA synthesis, and transcription of the progesterone receptor, lactoferrin, and glucose-6-phosphate dehydroge nase genes. Furthermore, serum levels of estradiol in the ERKO female are more than 10-fold higher than those in the wild type, consistent w ith a syndrome of hormone insensitivity.